title: Metal-sensitive and thermostable trypsin from the crevalle jack (Caranx hippos) pyloric caeca: purification and characterization.
abstract: BACKGROUND
  Over the past decades, the economic development and world population growth has led to increased for food demand. Increasing the fish production is considered one of the alternatives to meet the increased food demand, but the processing of fish leads to by-products such as skin, bones and viscera, a source of environmental contamination. Fish viscera have been reported as an important source of digestive proteases with interesting characteristics for biotechnological processes. Thus, the aim of this study was to purify and to characterize a trypsin from the processing by-products of crevalle jack (Caranx hippos) fish.
  
  
  RESULTS
  A 27.5 kDa trypsin with N-terminal amino acid sequence IVGGFECTPHVFAYQ was easily purified from the pyloric caeca of the crevalle jack. Its physicochemical and kinetic properties were evaluated using N-α-benzoyl-DL-arginine-p-nitroanilide (BApNA) as substrate. In addition, the effects of various metal ions and specific protease inhibitors on trypsin activity were determined. Optimum pH and temperature were 8.0 and 50°C, respectively. After incubation at 50°C for 30 min the enzyme lost only 20% of its activity. Km, kcat, and kcat/Km values using BApNA as substrate were 0.689 mM, 6.9 s-1, and 10 s-1 mM-1, respectively. High inhibition of trypsin activity was observed after incubation with Cd2+, Al3+, Zn2+, Cu2+, Pb2+, and Hg2+ at 1 mM, revealing high sensitivity of the enzyme to metal ions.
  
  
  CONCLUSIONS
  Extraction of a thermostable trypsin from by-products of the fishery industry confirms the potential of these materials as an alternative source of these biomolecules. Furthermore, the results suggest that this trypsin-like enzyme presents interesting biotechnological properties for industrial applications.
authors.affiliation: Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica (CCB) and Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Av, Prof, Moraes Rego s/n, Cidade Universitária, Recife, Pernambuco 50670-910, Brazil. ransoube@uol.com.br.
authors.forename: Helane Ms
authors.initials: HM
authors.lastname: Costa
authors.forename: Augusto Cv
authors.initials: AC
authors.lastname: Freitas Júnior
authors.forename: Ian Pg
authors.initials: IP
authors.lastname: Amaral
authors.forename: Izaura Y
authors.initials: IY
authors.lastname: Hirata
authors.forename: Patrícia Mg
authors.initials: PM
authors.lastname: Paiva
authors.forename: Luiz B
authors.initials: LB
authors.lastname: Carvalho
authors.forename: Vitor
authors.initials: V
authors.lastname: Oliveira
authors.forename: Ranilson S
authors.initials: RS
authors.lastname: Bezerra